lithium-chloride and acetylleucyl-leucyl-norleucinal

Incubation of rat hepatocytes with LiCl resulted in an overall increase in the activity ratio of glycogen synthase (GS), concomitantly with a decrease in active GS kinase-3 levels. GS total activity was also increased in a dose- and time-dependent manner. This latter effect correlated with the amount of immunoreactive enzyme determined by immunoblotting. Cycloheximide and actinomycin-D did not modify LiCl action on GS activity. Lithium ions did not induce any changes in GS mRNA levels. Furthermore, the increase in the total amount of GS induced by LiCl was further augmented after addition of a specific, calpain and proteasome inhibitor. Our results indicate that LiCl increases hepatocyte GS activity through increasing both the activation state of the enzyme and its cellular content. This latter increase is mediated through a modification of the proteasome-regulated proteolytic pathway of the enzyme.

Topics: Animals; Calpain; Cations, Monovalent; Cells, Cultured; Cycloheximide; Cysteine Proteinase Inhibitors; Dactinomycin; Enzyme Activation; Glycogen Synthase; Glycogen Synthase Kinase 3; Hepatocytes; Leupeptins; Lithium Chloride; Liver Glycogen; Male; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Synthesis Inhibitors; Rats; Rats, Sprague-Dawley; RNA, Messenger

The mammalian target of rapamycin is a serine-threonine kinase that regulates cell cycle progression. Rapamycin and its analogues inhibit the mammalian target of rapamycin and are being actively investigated in clinical trials as novel targeted anticancer agents. Although cyclin D1 is down-regulated by rapamycin, the role of this down-regulation in rapamycin-mediated growth inhibition and the mechanism of cyclin D1 down-regulation are not well understood. Here, we show that overexpression of cyclin D1 partially overcomes rapamycin-induced cell cycle arrest and inhibition of anchorage-dependent growth in breast cancer cells. Rapamycin not only decreases endogenous cyclin D1 levels but also decreases the expression of transfected cyclin D1, suggesting that this is at least in part caused by accelerated proteolysis. Indeed, rapamycin decreases the half-life of cyclin D1 protein, and the rapamycin-induced decrease in cyclin D1 levels is partially abrogated by proteasome inhibitor N-acetyl-leucyl-leucyl-norleucinal. Rapamycin treatment leads to an increase in the kinase activity of glycogen synthase kinase 3beta (GSK3beta), a known regulator of cyclin D1 proteolysis. Rapamycin-induced down-regulation of cyclin D1 is inhibited by the GSK3beta inhibitors lithium chloride, SB216763, and SB415286. Rapamycin-induced G1 arrest is abrogated by nonspecific GSK3beta inhibitor lithium chloride but not by selective inhibitor SB216763, suggesting that GSK3beta is not essential for rapamycin-mediated G1 arrest. However, rapamycin inhibits cell growth significantly more in GSK3beta wild-type cells than in GSK3beta-null cells, suggesting that GSK3beta enhances rapamycin-mediated growth inhibition. In addition, rapamycin enhances paclitaxel-induced apoptosis through the mitochondrial death pathway; this is inhibited by selective GSK3beta inhibitors SB216763 and SB415286. Furthermore, rapamycin significantly enhances paclitaxel-induced cytotoxicity in GSK3beta wild-type but not in GSK3beta-null cells, suggesting a critical role for GSK3beta in rapamycin-mediated paclitaxel-sensitization. Taken together, these results show that GSK3beta plays an important role in rapamycin-mediated cell cycle regulation and chemosensitivity and thus significantly potentiates the antitumor effects of rapamycin.

Topics: Aminophenols; Antibiotics, Antineoplastic; Antimanic Agents; Apoptosis; Breast Neoplasms; Cell Cycle; Cyclin D1; Cysteine Proteinase Inhibitors; Down-Regulation; Drug Resistance, Neoplasm; Female; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Half-Life; Humans; Indoles; Leupeptins; Lithium Chloride; Maleimides; Mitochondria; NF-kappa B; Paclitaxel; Proteasome Inhibitors; Sirolimus

In search of chemical substances applicable for the treatment of cancer and other proliferative disorders, we studied the signal transduction of Dictyostelium differentiation-inducing factors (DIFs) in mammalian cells mainly using HeLa cells. Although DIF-1 and DIF-3 both strongly inhibited cell proliferation by inducing G(0)/G(1) arrest, DIF-3 was more effective than DIF-1. DIF-3 suppressed cyclin D1 expression at both mRNA and protein levels, whereas the overexpression of cyclin D1 overrode DIF-3-induced cell cycle arrest. The DIF-3-induced decrease in the amount of cyclin D1 protein preceded the reduction in the level of cyclin D1 mRNA. The decrease in cyclin D1 protein seemed to be caused by accelerated proteolysis, since it was abrogated by N-acetyl-Leu-Leu-norleucinal, a proteasome inhibitor. DIF-3-induced degradation of cyclin D1 was also prevented by treatment with lithium chloride, an inhibitor of glycogen synthase kinase-3beta (GSK-3beta), suggesting that DIF-3 induced cyclin D1 proteolysis through the activation of GSK-3beta. Indeed, DIF-3 dephosphorylated Ser(9) and phosphorylated tyrosine on GSK-3beta, and it stimulated GSK-3beta activity in an in vitro kinase assay. Moreover, DIF-3 was revealed to induce the nuclear translocation of GSK-3beta by immunofluorescent microscopy and immunoblotting of subcellular protein fractions. These results suggested that DIF-3 activates GSK-3beta to accelerate the proteolysis of cyclin D1 and that this mechanism is involved in the DIF-3-induced G(0)/G(1) arrest in mammalian cells.

Topics: Animals; Cell Cycle; Cell Division; Cell Line; Cells, Cultured; Cyclin D1; Cyclin D2; Cyclin D3; Cyclins; Cysteine Endopeptidases; Dictyostelium; Electrophoresis, Polyacrylamide Gel; Endothelium, Vascular; G1 Phase; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; HeLa Cells; Hexanones; Humans; Leupeptins; Lithium Chloride; Microscopy, Fluorescence; Multienzyme Complexes; Phosphorylation; Proteasome Endopeptidase Complex; Resting Phase, Cell Cycle; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Messenger; Signal Transduction; Time Factors; Transfection; Umbilical Veins